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Immunoprecipitation

Juan S. Bonifacino1,  Esteban C. Dell'Angelica1,  Timothy A. Springer2

1National Institute of Child Health and Human Development, Bethesda, Maryland
2Center of Blood Research, Harvard Medical School, Boston, Massachusetts


Publication Name: 
Current Protocols in Immunology
Unit Number: 
UNIT 8.3
DOI: 
10.1002/0471142735.im0803s41
Print Publication Date: 
February, 2001
Online Posting Date: 
May, 2001
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Abstract

Immunoprecipitation consists of multiple ordered steps: lysing the cell with detergent if the antigen (usually a protein) to be precipitated is membrane-bound; binding of a specific antigen to an antibody; precipitating the antibody-antigen complex; washing the precipitate; and dissociating the antigen from the immune complex. The dissociated antigen is then analyzed by electrophoretic methods. In this unit, the basic protocol details the immunoprecipitation of a radiolabeled antigen with a specific antibody (polyclonal or monoclonal) covalently linked to Sepharose. Preparation of Ab-Sepharose is described in the Support Protocol. The first two alternate protocols present methods for precipitating or isolating the soluble immune complexes formed between a specific antibody and a radiolabeled antigen. Immunoprecipitation is achieved with polyclonal anti-immunoglobulin (Ig) serum, anti-Ig-Sepharose, Staphylococcus protein A or Streptococcus protein G bound to Sepharose, or Staphylococcus aureus bacteria which contain protein A on the cell surface. The third alternate protocol should be used for immunoprecipitation of antigens that are nonspecifically associated with other proteins. The fourth alternate protocol describes immunoprecipitation of unlabeled protein antigens with Ab-Sepharose.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution
  • Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution
  • Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions
  • Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent
  • Alternate Protocol 4: Immunoprecipitation Using Yeast Cells Disrupted with Glass Beads
  • Alternate Protocol 5: Immunoprecipitation with Antibody-Sepharose
  • Support Protocol: Preparation of Antibody-Sepharose
  • Alternate Protocol 6: Immunoprecipitation of Radiolabeled Antigen with Anti-Ig Serum
  • Basic Protocol 2: Immunoprecipitation-Recapture
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution

 Materials
  • Unlabeled or labeled cells in suspension
  • PBS (appendix 2A), ice cold
  • Nondenaturing lysis buffer (see recipe), ice cold
  • 50% (v/v) protein A–Sepharose bead (Sigma, Amersham Pharmacia Biotech) slurry in PBS containing 0.1% (w/v) BSA and 0.01% (w/v) sodium azide (NaN3)
  • Specific polyclonal antibody (antiserum or affinity-purified immunoglobulin) or monoclonal antibody (ascites, culture supernatant, or purified immunoglobulin)
  • Control antibody of same type as specific antibody (e.g., preimmune serum or purified irrelevant immunoglobulin for specific polyclonal antibody; irrelevant ascites, hybridoma culture supernatant, or purified immunoglobulin for specific monoclonal antibody; see Critical Parameters)
  • 10% (w/v) BSA
  • Wash buffer (see recipe), ice cold
  • Microcentrifuge with fixed-angle rotor (Eppendorf 5415C or equivalent)
  • Tube rotator (capable of end-over-end inversion)
  • Pasteur pipet attached to a vacuum trap

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer.

NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.


Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution

 Additional Materials (also see Basic Protocol 1)
  • Unlabeled or labeled cells grown as a monolayer on a tissue culture plate (unit 8.12)
  • Rubber policeman

NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions

 Additional Materials (also see Basic Protocol 1)
  • Denaturing lysis buffer (see recipe)
  • Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent)
  • 25-G needle attached to 1-ml syringe

Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent

 Additional Materials (also see Basic Protocol 1)
  • Detergent-free lysis buffer (see recipe)
  • 25-G needle attached to 3-ml syringe
    NOTE: All solutions should be ice-cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 4: Immunoprecipitation Using Yeast Cells Disrupted with Glass Beads

 Additional Materials (also see Basic Protocol 1)
  • Unlabeled or radiolabeled yeast cells
  • Lysis buffer, ice cold: nondenaturing, denaturing, or detergent-free lysis buffer (see recipes)
  • Glass beads (acid-washed, 425- to 600-µm diameter; Sigma)
NOTE: All solutions should be ice-cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 5: Immunoprecipitation with Antibody-Sepharose

 Materials
  • Unlabeled cells, surface-labeled cells (e.g., with 125I or biotin; unit 8.16) or biosynthetically 35S-, 3H-, or 14C-labeled cells (unit 8.12)
  • Triton X-100 lysis buffer (see recipe)
  • Dilution buffer (see recipe)
  • Antibody (Ab)-Sepharose (see Support Protocol)
  • Activated, quenched (control) Sepharose, prepared as for Ab-Sepharose (see Support Protocol) but eliminating Ab or substituting irrelevant Ab during coupling
  • Tris/saline/azide (TSA) solution (see recipe)
  • 0.05 M Tris×Cl, pH 6.8 (appendix 2A)
  • 2× SDS sample buffer (unit 8.4)

NOTE: Carry out all procedures in a 4°C cold room or on ice.

Support Protocol: Preparation of Antibody-Sepharose

 Materials
  • 1 to 30 mg/ml antigen-specific monoclonal or polyclonal antibody
  • 0.1 M NaHCO3/0.5 M NaCl
  • Sepharose CL-4B (or Sepharose CL-2B for high-molecular-weight antigens; Amersham Pharmacia Biotech)
  • 0.2 M Na2CO3
  • Cyanogen bromide (CNBr)/acetonitrile (see recipe)
  • 1 mM and 0.1 mM HCl, ice-cold
  • 0.05 M glycine (or ethanolamine), pH 8.0
  • Tris/saline/azide (TSA) solution (see recipe)
  • Dialysis tubing (molecular weight cutoff >10,000)
  • Whatman no. 1 filter paper
  • Buchner funnel
  • Erlenmeyer filtration flask
  • Water aspirator

Alternate Protocol 6: Immunoprecipitation of Radiolabeled Antigen with Anti-Ig Serum

 Additional Materials (also see Alternate Protocol 5)
  • Normal serum
  • Anti-Ig serum (Zymed Laboratories)
  • Antigen-specific antiserum or antigen-specific purified monoclonal antibody or antigen-specific hybridoma culture supernatant

Basic Protocol 2: Immunoprecipitation-Recapture

 Materials
  • Elution buffer (see recipe)
  • Beads containing bound antigen (see Basic Protocol 1, step )
  • 10% (w/v) BSA
  • Nondenaturing lysis buffer (see recipe)
  • Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 8.3.1
    Schematic representation of the stages of the immunoprecipitation protocol presented in Basic Protocol 1. (1) Cell lysis: antigens are solubilized by extraction of the cells in the presence or absence of detergents. To increase specificity, the cell lysate can be precleared with protein A–agarose beads (steps 15 to 17, not shown). (2) Antibody immobilization: a specific antibody is bound to protein A–agarose beads. (3) Antigen capture: the solubilized antigen is isolated on antibody-conjugated beads.

    View Image
  • Figure 8.3.2
    Schematic representation of the stages of the immunoprecipitation protocols using either antibody-Sepharose (left, see Alternate Protocol 5) or anti-Ig serum (right, see Alternate Protocol 6). (1) Cell lysis. (2) Immunoprecipitation using specific antibodies coupled covalently to Sepharose beads (left) or specific antibodies combined with anti-Ig serum (right). (3) Washing. (4) Dissociation of the antigen-antibody complex in sample buffer for electrophoresis.

    View Image
  • Figure 8.3.3
    Scheme showing the stages of immunoprecipitation-recapture. (1) Dissociation and denaturation of the antigen: an antigen immunoprecipitated with antibody 1 bound to protein A–agarose beads is dissociated and denatured by heating in the presence of SDS and DTT. (2) Immobilization of the second antibody: antibody 2 is bound to protein A–agarose beads. (3) Recapture: the denatured antigen 2 (striped oval) is recaptured on antibody 2 bound to protein A–agarose beads. Alternatively, antibody 1 can be used again for further purification of the original antigen (square).

    View Image
  • Figure 8.3.4
    Example of an immunoprecipitation-recapture experiment. Human M1 fibroblasts were labeled overnight with [35S]methionine (unit 8.12) and extracted with nondenaturing lysis buffer (see Basic Protocol 1). The cell extract was then subjected to immunoprecipitation with antibodies to BSA (irrelevant antibody control; lane 1) and to the AP-3 adaptor (3; lane 2), a protein complex involved in protein sorting. Notice the presence of several specific bands in lane 2. The AP-3 immunoprecipitate was denatured as described in Basic Protocol 2 and individual components of the AP-3 complex were recaptured with antibodies to two of its subunits: 3 (Mr ~22,000; lane 3) and µ3 (Mr~47,000; lane 4). An immunoprecipitation with an antibody to BSA was also performed as a nonspecific control (lane 5). The amount of immunoprecipitate loaded on lanes 1 and 2 is ~ 1/10 the amount loaded on lanes 3 to 5. Notice the presence of single bands in lanes 3 and 4. The positions of Mr standards (expressed as 10–3 × Mr) are shown at left. IP, immunoprecipitation.

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  • Figure 8.3.5
    Lowering background by washing with SDS and sodium deoxycholate (DOC). In this experiment, BW5147 cells (mouse thymoma) labeled with [35S]methionine for 1 hr were extracted with nondenaturing lysis buffer (see Basic Protocol 1). The extracts were subjected to immunoprecipitation with protein A–agarose beads incubated with either preimmune (PI) or immune (I) serum from a rabbit immunized with the ribosomal protein L17 (doublet at Mr ~22,000). Lanes 1 and 2 correspond to immunoprecipitates obtained using the protocols described in this unit. Notice the presence of nonspecific bands and/or associated proteins in lane 2. Lanes 3 and 4 correspond to beads that were washed an additional time with 0.1% (w/v) SDS and 0.1% (w/v) DOC. Notice the disappearance of most of the nonspecific bands and/or associated proteins. The positions of Mr standards (expressed as 10–3 × Mr) are shown at left.

    View Image

Literature Cited

 Literature Cited
    Anderson, D.J. and Blobel, G. 1983. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 96:111-120.
    Aruffo, A. 1998. Transient expression of proteins using COS cells. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.13.1-16.13.7. John Wiley & Sons, New York.
    Cuatrecasas, P. 1970. Protein purification by affinity chromatography. J. Biol. Chem. 245:3059.
    Earl, P.L. and Moss, B. 1998. Characterization of recombinant vaccinia viruses and their products. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.18.1-16.18.11. John Wiley & Sons, New York.
    Earl, P.L., Cooper, N., Wyatt, L.S., Moss, B., and Carroll, M.W. 1998a. Preparation of cell cultures and vaccinia stocks. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.16.1-16.16.13. John Wiley & Sons, New York.
    Earl, P.L., Moss, B., Wyatt, L.S., and Carroll, M. 1998b. Generation of recombinant vaccinia virus. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.17.1-16.17.19. John Wiley & Sons, New York.
    Franzusoff, A., Rothblatt, J., and Schekman, R. 1991. Analysis of polypeptide transit through yeast secretory pathway. Methods Enzymol. 194:662-674.
    Gelb, W.G. 1973. Affinity chromatography: For separation of biological materials. Am. Lab. 81:61-67.
    Gersten, D.M. and Marchalonis, J.J. 1978. A rapid, novel method for the solid-phase derivatization of IgG antibodies for immune-affinity chromatography. J. Immunol. Methods 24:305-309.
    Harford, J. 1984. An artefact explains the apparent association of the transferrin receptor with a ras gene product. Nature 311:673-675.
    Harlow, E. and Lane, D. 1999. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    Irving, R.A., Hudson, P.J., and Goding, J.W. 1996. Construction, screening and expression of recombinant antibodies. In Monoclonal Antibodies: Principles and Practice (J.W. Goding, ed.). Academic Press, London.
    Kessler, S.W. 1975. Rapid isolation of antigens from cells with a staphylococcal protein A–antibody adsorbent: Parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115:1617.
    Köhler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predetermined specificity. Nature 256:495-497.
    March, S.C., Parikh, I., and Cuatrecasas, P. 1974. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60:149-152.
    Moss, B. and Earl, P.L. 1998. Expression of proteins in mammalian cells using vaccinia viral vectors. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.15.1-16.15.5. John Wiley & Sons, New York.
    Nisonoff, A. 1984. Introduction to Molecular Immunology. Sinauer Associates, Sunderland, Mass.
    Rapley, R. 1995. The biotechnology and applications of antibody engineering. Mol. Biotech. 3:139-154.
 Key References
    Harlow and Lane, 1988. See above.
    Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56:734-749.
    Hjelmeland, J.M. and Chrambach, A. 1984. Solubilization of functional membrane proteins. Methods Enzymol. 104:305-318.

The above references describe various detergents and solubilization conditions.

     
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